This document discusses luminscence immunoassay and chemiluminescence immunoassay (CLIA) specifically. It begins with an introduction to luminescence and the different types. It then explains the principle of CLIA, describing it as an immunoassay that uses a chemiluminescent probe to label antibodies. The document outlines the different types of CLIA, including direct, indirect, and sandwich assays. It discusses applications of CLIA in estimating analytes like hormones, tumor markers, and COVID-19 markers. Finally, it covers the advantages and disadvantages of CLIA.

1. LUMINOSCENCE
IMMUNOASSAY
SUBMITTED BY- SK AZIZ UDDIN
SUBMITTED TO- DR.RIKESHWAR PRASAD
DEWANGAN
COURSE-M.PHARM
BRANCH-PHARMACEUTICAL ANALYSIS
COLLEGE NAME- JAMIA HAMDARD
YEAR-2020-2022

2. CONTENTS
INTRODUCTION (LUMINESCENCE)
TYPES OF LUMINESCENCE.
PRINCIPLE( CHEMILUMINESCENCE)
PRINCIPLE OF CLIA
TYPES OF CLIA
APPLICATION OF CLIA
ADVANTAGES & DISADVANTAGES OF CLIA
REFERENCES

3. INTRODUCTION
 Luminescence refers to the release of
energy from an electronically excited
molecule in the form of visible light.
When the excited molecule returns to
the ground state it releases a photon
of light. The catch-all title of
luminescence covers a range of
detection systems, including
chemiluminescence,
phosphorescence, bioluminescence
and fluorescence.
 Incandescence, which is light emitted

4. LUMINESCENCE
 Cold light that can be emitted at lower
temperature.
 Source kicks an electron of an atom
out of its lowest energy ‘ground’ state
higher energy ‘excited’ state.
 Finally electron returns the energy in
the form of light so it can fall back to
its ‘ground’ state.

6. TYPES LUMINESCENCE
Excitation
event
process
Chemicals Luminol Isoluminol acridinium
ester
Chemiluminesc
en-ce
Biochemical Luciferin aequorin Bioluminescenc
e
Electromagnetic Ruthenium Tris (bipyridly)
chelate
Electroluminesc
ence
Photons Inorganic phosphors Photoluminesce
nce

7. CHEMILUMINESCENCE
 Emission of light with limited emission of
heat , as the result of a chemical
reaction.
 [A]+[B]→[◊]→[products]+ light
 Where ;- [A], [B] ; reactants
[◊] is excited intermediate
 Commonly used catalysts in this reaction are
 Alkaline phosphate(ALP) label is 1,2 dioxane
 Horse radish peroxidase(HRP) label is luminol.
 Metal ions complexes (copper and iron
phthalocyanine complex)
-15

8. CONTINUE;-
 For example if [A] is luminol and [B] is
hydrogen peroxide in the presence of
a suitable catalyst we have:
 Luminol+ H2 O2 → 3-APA[◊]→3-APA +
light
 Where
3-APA is 3- amino phthalate
3-APA[◊] is the excited state producing light as
it decays to lower energy level.

9. CONTINUE;-

10. Application of
chemiluminescence
 Chemiluminescence immunoassay
 DNA hybridization detection
 Western blotting
 Forensic science
 Food analysis

11. CHEMILUMINESENCE
IMMUNOASSAY(CLIA)
 Principle-: CLIA utilize chemical probe which
could generate light emission through
chemical reaction to label the antibody. It is
an assay that combine chemiluminescence
technique with immunochemical reactions.
Similar with other labelled
immunoassays(RIA, FIA, ELISA).etc
 Provides a sensitive , high throughput
alternative to conventional colorimetric
methodologies.
 Uses chemiluminescent substrate, hydrogen
peroxide , enhancers
 Stopping reagent is not required.
 Incubation period is small

12. Types of chemiluminescent
assay.
 Direct chemiluminescent assay
 In this method luminophore markers used are
acridinium and ruthenium esters. This kind of
chemical with special structure can transfer to
an excited state through chemical reaction.
Exposure of an acridinium ester label to an
alkaline hydrogen peroxide solution triggers a
flash of light. A subsequent development has
been the acridinium sulfonamide ester labels.
It is also triggered by alkaline hydrogen
peroxide to emit a flash of light. The light
emission mechanism of acridinium ester is
shown in Figure

13. Direct chemiluminescent
assay

14. Indirect chemiluminescent
assay
 Enzymatic markers used indirect methods
are alkaline phosphates with adamantly 1,2
dioxetane aryl phosphate(AMPPD) and horse
radish peroxidase(HRP) each has its own
luminescent substrates.
 Luminol is a very common chemiluminescent
substrate used for detection of HRP. HRP
catalyzes the decomposition of luminol in the
presence of peroxide to produce an excited
state intermediate. Flashes of visible light
(maximum at 425nm) is emitted on decay of
the singlet intermediate.

15. Indirect chemiluminescent
assay
 AMPPD is a derivative of 1, 2-
dioxetane substrates. It has a similar
mechanism of chemiluminescence.
On enzymatic cleavage of the
phosphate group, this compound
becomes destabilized and
decomposes via an intermediate
anion, AMPD, which is moderately
stable. The wavelength of maximum
light emission is 470nm.

17. Sandwich chemiluminescent
immunoassay
 Sandwich CLIA is a kind of detection method combined
double antibody sandwich method with
chemiluminescence detection method. It regularly
includes lots of steps, which are shown below. At first,
the microtiter plate has been pre-coated with an
antibody specific to analyte. Then, standards or
samples are added to the appropriate microtiter plate
wells, the analyte in standards and samples will bound
to the immobilized Ab. Next, biotin-conjugated antibody
is added and binds to the analyte absorbed on the
plate. The complex of two antibodies and analytes in
the wells act as a “sandwich” structure. After the
unbound biotin-conjugated antibody is washed away,
avidin-conjugated Horseradish Peroxidase (HRP) is
added to each micro plate well, after incubation, luminol
is added into the micro wells, and then relative
luminosity values (RLU) will be scanned by photon

19. USES
 Used to estimate analytes which have extremely low
conc. In the blood such as hormones, serological
markers and drugs.
 Hormones ; insulin, thyroxin,
estradiol,testosterone,progesterone,prolactin, LH, FSH
etc. Vitamin; vit B12
 Tumor markers: bone morphogenic protein-2, carcino
embryonic antigen(CEA), alpha fetoprotein(AFP)
 Also COVID-19 marker, biochemical marker is also
detected
 Human beta chorionic gonadotropin
 C- reactive protein.
 Tumor necrosis factor
 To detect ATP specific enzymes.(Luciferase,creatin

20. ADVANTAGES OF CLIA
 It is a analytical methods reside in the
wide dynamic range.
 It has high signal intensity, absence of
interfering emissions(i.e. High
specificity)
 Rapid acquisition of the analytical
signal.
 High stability of reagents and their
conjugates.
 Low consumption of reagents.
 Random access, reduced incubation

21. DISADVANTAGES OF CLIA
Light leaks from assay reagent &
reaction vessels
Ultra sensitive- stringent controls on
purity of reagents
High intensity light emission leads to
pulse pile up in photomultiplier tubes
leads to underestimation.
Limited Ag detection
High costs
Limited tests panel

22. REFERENCES
 Internet browsing (GOOGLE)
 http://www.cloud-
clone.com/topic/201511200859289789.htm#:~:text=San
dwich%20CLIA%20is%20a%20kind,method%20with%2
0chemiluminescence%20detecion%20method.&text=Th
e%20complex%20of%20two%20antibodies,as%20a%2
0%E2%80%9Csandwich%E2%80%9D%20structure.
 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5483212
/
 You tube video
 Google Slide share
 Immunoassay book.

23. THANK YOU