Chemiluminescence

Immunoassay (CLIA) Technique

Tapeshwar Yadav
(Lecturer)
BMLT, DNHE,
M.Sc. Medical Biochemistry
Background:
 Immunoassay (IA) as an analytical technique is widely applied
in clinical chemistry, bioanalysis, toxicological analysis,
pharmaceutical analysis, and environmental analysis, due to its
high sensitivity, high selectivity, rapid detection and possible
analysis of difficult matrices without extensive pre-treatment.
 Radioimmunoassay (RIA) has been developed since Yalow
and Berson introduced it in 1959 by using 125I as a label. Although
RIA methods are reliable and accurate, they suffer from the
problems associated with radioisotopes, which restrict their use to
specialized laboratories. They also suffer from the drawback of the
short half-life using 125I as a label
Contd…
 Enzyme-linked immunosorbent assays (ELISAs) are
among the most extensively used types of IA and are safer
and easier than the RIA.
ELISA could be based on colorimetric, fluorescence or
Chemiluminescence (CL) detection. However, the
sensitivity of conventional colorimetric detection is relatively
low.
IA has been applied to develop assays with very high
sensitivity detection, which strongly depends on the affinity
of specific antibodies (Abs) and on the sensitivity of the
detection method.
Contd…
 Among assay methods, Chemiluminescence (CL) detection
represents a versatile, ultrasensitive tool with a wide range of
applications in biotechnology. It also gives a sensitive, rapid
alternative to radioactivity as a detection principle in IA for the
determination of molecules (e.g., proteins, hormones, drugs,
nucleic acids and environmental pollutants). CL is now commonly
used for IA in the form of a CL label or as a CL detection reaction
for an enzyme or a nanoparticle (NP) label.
 In recent years, CLIA has become very popular in
clinical chemistry and environmental analysis, due to its high
sensitivity, wide dynamic range and complete automation.
With the development and application of recombinant Ab (rAb)
technology, markers and related techniques, solid-phase materials
and improvements in automation, integration and miniaturization,
CLIA has acquired an entirely new appearance.
LUMINESCENCE

 “ Cold light” that can be emitted at lower temperature

 Source kicks an electron of an atom out of its lowest
energy “ground” state into a higher energy “excited” state
 Finally electron returns the energy in the form of light so
it can fall back to its “ground” state
TYPES LUMINESCENCE
Excitation event process

Chemicals Luminol Isoluminol Chemiluminescence

acridinium ester

Biochemical Luciferin Bioluminescence

aequorin

Electromagnetic Ruthenium Electroluminescence

Tris (bipyridly) chelate

Photons inorganic phosphors Photoluminescence

CHEMILUMINESCENCE
Emission of light with limited emission of heat
(luminescence), as the result of a chemical reaction.
[A] + [B] → [◊] → [Products] + light

[A], [B]: reactants

[◊]: excited intermediate
For example, if [A] is luminol and [B] is hydrogen peroxide
in the presence of a suitable catalyst we have:
luminol + H2O2 →3-APA[◊] →3-APA + light
Where:
3-APA is 3-aminophthalate
3-APA[◊] is the excited state producing light as it decays
to a lower energy level.
CHEMILUMINISCENCE

Luminol and peroxidase

before adding H2O2

Chemiluminiscence after
addition H2O2
Application of Chemiluminiscence

 Chemiluminesence immunoassay
 DNA hybridization detection
 Western blotting
 Forensic science
 Food analysis
Chemiluminescence
Immunoassay (CLIA) Technique
 Introduction:
 Chemiluminescence Immunoassay (CLIA) using Microplate
luminometers provides a sensitive, high throughput, and economical
way to quantitatively measure antigen in cell lysates, plasma, urine,
saliva, tissue and culture media samples.
 Chemiluminescence Immunoassay does not require long incubations
and the addition of stopping reagents, as is the case in conventional
colorimetric assays such as Enzyme-linked ImmunoSorbent Assays
(ELISA).
 Among various enzyme assays that employ light-emitting reactions,
one of the most successful assays is the enhanced chemiluminescent
immunoassay involving a horseradish peroxidase (HRP) labeled
antibody or antigen and a mixture of chemiluminescent substrate,
hydrogen peroxide, and enhancers.
 Contd…

CLIA are designed to detect glow-based chemiluminescent

reactions.
This provide a broader dynamic assay range, superior low-end
sensitivity, and a faster protocol than the conventional Enzyme-
linked immunosorbent assays (ELISA).
It covers Thyroid function markers, Gonadal hormones, Tumor
markers, Diabete marker, Cardiac marker and other markers.
They can be used to replace conventional colorimetric ELISAs
that have been widely used in many research and diagnostic
applications.
 CHEMILUMINESCENCE
IMMUNOASSAY
(CLIA)

Provides a sensitive, high throughput

alternative to conventional colorimetric
methodologies.

Principle: -same as ELISA

-uses chemiluminescent substrate,
hydrogen peroxide, enhancers
-stopping reagent is not required
-Incubation period is small
 Principle:
 In the presence of complimentary antigen and antibody, the paratope
of the antibody binds to the epitope of the antigen to form an Ag-Ab
or an immune complex.
 Estimating the levels of such immune complex by use of labeled
antibodies form the basis of CLIA.
 It involves use of stationary solid particles coated either with the Ag
or Ab of interest.
 Post incubation, which ensures intact immune complexes are
formed, substrate is added.
 This results in generation of light, the intensity of which is directly
proportional to the amount of labeled complexes present and which
indirectly aids in quantification of the analyte of interest.
 The intensity of light is measured in terms of Relative Light Units
(RLU).
 Monoclonal antibody coated well

Test specimen (serum)

HRP labelled antibody conjugate

Test antigen: sandwich between solid phase Ab and enzyme

labelled Ab
 Incubate for 1 hr at 37° C

Remove unbound enzyme labeled Ab

Chemiluminescence reagent added

Read relative light unit with luminometer

Principle:
 Labels:
 Reagents required for reactions that produce CL may be coupled to Abs
or antigens (Ags) and used as labels for IA. Since the first report on CL
labels in 1976
 This category involves labels that are consumed in the CL analytical
reaction (e.g., luminol derivatives, acridinium esters and NPs).
 Luminol is the best known and one of the most efficient CL reagents. It
is coupled to ligands via reactions involving the amino group.
 However, the resulting conjugates have lower CL efficiencies than the
parent compounds. Labels derived from isoluminol have been more
successful.
 
 Solid-phase materials:
 Commonly used solid-phase are 96-well microtitrationplates prepared
with polystyrene. For the purposes of IA, the microplates are pre-coated
with capturing protein like Ab to allow analyte immobilization.
Instrumentation:
• USES
 Hormones: insulin, thyroxin, estradiol, testosterone,
progesterone
 Vitamin: vit B12
 Tumor markers: bone morphogenic protein-2, carcino
embryonic antigen (CEA), alpha fetoprotein (AFP)
 Human beta chorionic gonadotropin
 C-reactive protein
 Tumor necrosis factor
 DNA hybridization detection
Southern blotting Involves DNA separation, transfer and
hybridization

Hybridization - Process of forming a double-stranded DNA

molecule between a single-stranded DNA probe and a single-
stranded target DNA
3.The restriction fragments
present in the gel are
denatured with alkali and
transferred onto a
nitrocellulose filter or
nylon membrane by
blotting.
 This procedure preserves
the distribution of the
fragments in the gel,
creating a replica of the gel
on the filter.
4. The filter is incubated under
hybridization conditions with a
specific HRP labelled DNA probe.
The probe hybridizes to the
complementary DNA restriction
fragment. 
Detection reagent containing
H2O2 & luminol is added onto the
membrane
USES
 Identifying DNA in crime case
 Paternal Dispute
 Classify DNAs of various organism
Steps in southern blotting

1.The DNA to be analyzed,

such as the total DNA of an
organism, is digested to
completion with a restriction
enzyme.

2.The complex mixture of

fragments is subjected to gel
electrophoresis to separate
the fragments according to
size.
Western blotting
Western blotting (or protein immunoblotting) is a technique
widely used to detect specific proteins in samples of tissue
homogenate, cell lysates, cell culture supernatants or body fluids.
Western blotting
Forensic science
Chemiluminescence is used by criminalists to detect
traces of blood at crime scene

Solution: luminol powder (C8H7O3N3), hydrogen

peroxide, and a hydroxide (eg. KOH) sprayed
where blood might found

Tiny amount of iron from Hb in blood serves as

catalyst for the chemiluminescence reaction,
luminol to glow
A trail of blood made visible with the use of the
reagent luminol.
Food analysis
Organophosphorous most popular pesticide

Most commonly used organophosphorous: QUINALPHOS

(O,O diethyl-o-quinoxalinly phosphorothioate)
Food analysis

Quinalphos +H2O2 peroxophosphonate

Peroxophosphonate+ luminol 3aminophthalate

anion*

3-aminophthalate* 3-
aminophthalate
+
observed
emission
Limitations
•Light leaks from assay reagent & reaction vessels
•Ultra sensitive – stringent controls on purity of reagents
•High intensity light emission leads to pulse pileup in
photomultiplier tubes leads to underestimation