3. CONTENTS
ELISA
Introduction to ELISA
Principle & procedure
Materials needed
Types of ELISA
Advantages & disadvantages of ELISA
Applications
RIA
Introduction to RIA
Principle & procedure
Materials needed
Advantages & disadvantages of RIA
Instrumentation
Applications
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4. INTRODUCTION TO ELISA
ELISA, or enzyme-linked immunosorbent assay,
are quantitative immunological procedures in
which the Ag- Ab reaction is monitored by
enzyme measurements.
The term ELISA was first used by Engvall &
Perlma in 1971.
The ELISA test, or the enzyme immunoassay
(EIA), was the first screening test commonly
employed for HIV. It has a high sensitivity.
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5. Why known as ......?
Enzyme Linked Immunosorbent Assay
Antigen of interest is absorbed on to plastic. 1
’(.surface (‘sorbent
Antigen is recognised by specific antibody .2
’(.(‘immuno
This antibody is recognised by second antibody .3
(‘immuno’( which has enzyme attached (‘enzyme-
’(.linked
Substrate reacts with enzyme to produce product, .4
. usually coloured 5
6. BASIC PRINCIPLE OF ELISA
Use an enzyme to detect the binding of
antigen (Ag) antibody (Ab).
The enzyme converts a colorless substrate
(chromogen) to a colored product,
indicating the presence of Ag : Ab binding.
An ELISA can be used to detect either the
presence of Antigens or antibodies in a
sample depending how the test is designed.
ELISA was dveloped in 1970 and became
rapidly accepted
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9. ANTIGEN (Ag)
Any molecule that induces production of
antibodies when introduced in the body of an
animal is called antigen.
OR
any “thing”, foreign to the immune system. e.g.
bacteria, viruses, (or their parts), pollen, etc.
Protein molecule
Carbohydrate molecule. SYMBOL FOR ANTIGEN
Microorganisms
Allergens.
Viruses Etc.
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10. ANTIBODY ( Ab)
Antibody: proteins produced by the
immune system which help defend
against antigens
SYMBOL FOR
ANTIBODY
Y
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12. Specimen Sample For ELISA
SERUM
CSF
SPUTUM
URINE
SEMEN
SUPERNATANT OF CULUTRE
STOOL
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13. Enzymes Used in Elisa
Horseradish peroxidase (most commonly
used)
Alkaline Phosphatase
β-galactosidase
Lactoperoxidase
Tetra Methyl benzidine
In case of peroxidase, the substrate hydrogen peroxide is converted
into water and o2 in the presence of electron donors . (like
diaminobenzidine or 4-chloronaphthol which themselves oxidized in
the reaction).
Oxidation of diaminobenzidine produces dark brown color while that
of 4-chlorornaphthol yields purple color which is the basis of ELISA
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14. ENZYME SUBSTRATE
Initially
the substrate should be colorless
After degradation by the enzyme it
should be strongly colored or fluorescent.
ENZYME SUBSTRATE CHROMOGEN STOPPING
Alkaline p-NPP p-NPP+ 1 M NaOH
Phosphatase diethandamine+Mg
Cl2
Horse radish H2O2 Tetramethylbenzidi 1 M H2SO4
Peroxidase ne + Phosphate –
Citrate buffer
Horse radish H2O2 O– 1 M HCl
Peroxidase Phenylenediamine
+ HCl
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15. Secondary
antibody
Substrate
e
Enzym
Coloured
product
Primary
antibody
Different antigens in sample
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19. Sandwich elisa
Antigens such as tumor markers, hormones and
serum proteins may be determined
Antigen in the sample binds with the capture antibody
on the microwell and becomes immobilized.
The antibody of the enzyme conjugate binds with the
immobilized antigen to form a sandwich of antibody-
antigen-antibody/enzyme bound to the microwell.
Enzyme reaction product is directly proportional to
concentration of standard or analytical antigen 19
21. ELISA SANDWICH FORMAT
YYY
Antibody
YYY
2nd antibody with enzyme
Y YYY
enzyme produces colour
Y YYY
Y
YYY
Antibody/Antigen
YYY
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22. Competitive Elisa
Used to determine small molecule antigens.(T3,T4,progesterone etc.)
antibody coated microwell
serum antigen and labelled antigen added together--competition.
antibody-antigen-enzyme complex bound is inversely related to the concentration
of antigen present in the sample.
The bound enzyme conjugate reacts with the chromogenic substrate added to
produce a color reaction (blue to yellow color). .
Increased serum antigen results in reduced binding of the antigen-enzyme
conjugate with the capture antibody producing less enzyme activity and color
(yellow) formation
Substrate product concentration is inversely proportional
to the concentration of standard or test antigen added
22
23. ( to detect Ab (HIV, HCV
( to detect Ag ( Tumor Markers, Hormones
( to detect Ag ( Free Testosterone
Comparison between Indirect Sandwich & Competitive ELISA
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25. Importance of incubation step:-
During the test performance incubation
time and mentioned temperature is must
required For the proper binding between
antigen and antibody and also binding with
conjugate and color development of
substrate.
Importance of Washing :- For the removal
of any unbound Antibody/Antigen proper
washing and taping is required other wise
we get the incorrect result.
So incubation & washing is much important
for good results.
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26. Elisa Plate
Microtitre wells
Generally 96
wells
Marked on one
side alphabetically
Numerically on
the other side
Comes with the
kit
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27. TEST PERFORMANCE
Using a clean
Pipette , add 100
µL of diluted
serum sample
(Dilute the sera to
be tested 1:100 in
the sample
diluents) to each
.well
Incubate 1 hour
.at 37°C
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29. ELISA PLATE READY
FOR READING
Measures the absorbance at
450nm With the help of
ELISA READER.
Calculate the absorbance
for each sample and
reference.
We used Ascent Software
for Calculation of the result
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30. Advantages of ELISA
Reagents are relatively cheap & have a long
shelf life
ELISA is highly specific and sensitive
No radiation hazards occur during labelling
or disposal of waste.
Easy to perform and quick procedures
Equipment can be inexpensive and widely
available.
ELISA can be used to a variety of infections.
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31. Disadvantages of ELISA
Measurement of enzyme activity can be more
complex than measurement of activity of some
type of radioisotopes.
Enzyme activity may be affected by plasma
constituents.
Kits are commercially available, but not cheap
Very specific to a particular antigen. Won’t
recognize any other antigen
False positives/negatives possible, especially with
mutated/altered antigen
31
32. Limitations
Results may not be absolute •
Antibody must be available •
Concentration may be unclear •
False positive possible •
False negative possible •
32
33. APPLICATIONS OF ELISA
detection of Mycobacterium antibodies in tuberculosis
detection of rotavirus in feces
detection of hepatitis B markers in serum
detection of HIV antibodies in blood samples
It has also found applications in the food industry in detecting potential
eggs food allergens, such as milk, peanuts, walnuts, almonds, and
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34. APPLICATIONS OF ELISA
1- Hormones 7- Vaccine Quality Control
2- Proteins 8- FOR GMO (Genetically modified
organism)
3- Infectious Agent ( Viral, Bacterial, 9- For Rapid Test
Parasitic, Fungal )
4- Drug Markers 10- IgG, IgM, IgA
5- Tumor Markers 11- In New Born Screening
6- Serum Proteins 12- In Clinical Research
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40. INTRODUCTION
Radioimmunoassay (RIA) is a very sensitive in vitro assay technique
used to measure concentrations of antigens (for example, hormone levels in
theblood) by use of antibodies.
The RAST test (radioallergosorbent test) is an example of
radioimmunoassay. It is used to detect the causative allergen for
an allergy
To perform a radioimmunoassay, a known quantity of an antigen is
made radioactive, frequently by labeling it with gamma-
radioactive isotopes of iodine attached to tyrosine.
40
41. To perform a radioimmunoassay, a known quantity of an antigen is
made radioactive, frequently by labeling it with gamma-
radioactive isotopes of iodine attached to tyrosine.
This radiolabeled antigen is then mixed with a known amount
of antibody for that antigen, and as a result, the two specifically bind to one
another.
Then, a sample of serum from a patient containing an unknown quantity of
that same antigen is added.
This causes the unlabeled (or "cold") antigen from the serum to compete
with the radiolabeled antigen ("hot") for antibody binding sites. As
theconcentration of "cold" antigen is increased, more of it binds to the
antibody, displacing the radiolabeled variant, and reducing the ratio of
antibody-bound radiolabeled antigen to free radiolabeled antigen. The
bound antigens are then separated from the unbound ones, and the
radioactivity of the free antigen remaining in the supernatant is measured
using a gamma counter.
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42. Principle of Radioimmunoassay
Principle:Uses an immune reaction
[Antigen – Antibody reaction] to estimate
a ligand
Ag + Ag* + Ab AgAb + Ag*Ab + Ag +
Ab*
◦ Unbound Ag* and Ag washed out
◦ Radioactivity of bound residue measured
◦ Ligand conc is inversely related to radioactivity
[Ag : ligand to be measured ; Ag* radiolabelled
ligand]
42
43. Advantages &
Disadvantages of RIA
Advantages
◦ Highly specific: Immune reactions are specific
◦ High sensitivity : Immune reactions are
sensitive
Disadvantages
◦ Radiation hazards: Uses radiolabelled reagents
◦ Requires specially trained persons
◦ Labs require special license to handle
radioactive material
◦ Requires special arrangements for
Requisition, storage of radioactive material
radioactive waste disposal.
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44. Requirements for RIA
1. Preparation & characterisation
of the Antigen [Ligand to be
analysed]
2. Radiolabelling of the Antigen
3. Preparation of the Specific
Antibody
4. Development of Assay System
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45. Preparation & Radiolabelling of
the Antigen
Antigens prepared by..
◦ Synthesis of the molecule
◦ Isolation from natural sources
Radiolabelling [Tagging procedure]
◦ 3 H 14 C 125 I are used as radioactive tags
◦ Antigens are tagged to 3 H 14 C 125
◦ Tagging should NOT affect Antigenic
specificity & Antigenic activity !
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46. Preparation of the Specific
Antibody
Antigen injected intradermally into rabbits or
guinea pigs antibody production
Antibodies recovered from the serum
Some ligands are not Antigenic
◦ Hormones, Steroids, Drugs HAPTENS
◦ Eg: Gastrin, Morphine,
◦ Haptens conjugated to albumin antigenic
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47. Development of the Assay System
A crucial step is separation of unbound
antigens
This achieved by binding the antibodies to the
microtitre well surface [Solid phase RIA]
Antigens bound to the fixed antibodies remain
stuck to the inner surface
Decanting & washing the well removes
unbound antigens
Other techniques of separation: Centrifugation
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48. Assay Procedure
Add known amounts of the test sample + labelled
antigen into the microtitre wells
Incubate allow the reaction to reach completion
Decant & wash contents of the well removes all
unbound antigens
Radioactivity remaining in the Microtitre wells
measured by a Counter [GM counter , Scintillation
counter etc]
Intensity of radioactivity is inversely correlated with
the conc of antigens in the test sample
Sensitive to very low conc of antigens
48
51. Advantages
Radioimmunoassay is widely-used
because of its great sensitivity.
Using antibodies of high affinity, it is
possible to detect a few picograms
(10−12 g) of hormone in the tube.
The greater the specificity of the
antiserum, the greater the specificity
of the assay
51
52. limitations
The main drawbacks to radioimmunoassay are
the expense and hazards of preparing and
handling the radioactive antigen.
Both 125I or 131I emit gamma radiation that requires
special counting equipment
The body concentrates iodine atoms —
radioactive or not — in the thyroid gland where
they are incorporated in thyroxine (T4).
52
54. INSTRUMENTATION
Radiation will hit silver grains in emulsion and expose them
Expose to film
or emulsion
Isotope will emit
(radiation (usually beta
Incubate tissue with
radioactive ligand
Autoradiography
54
56. REFERENCES
Engvall E, Perlman P (1971(. "Enzyme-linked immunosorbent assay (ELISA(.
Quantitative assay of immunoglobulin G". Immunochemistry 8 (9(: 871–4.
Gofflot; El (2004(.
Journal of Immunoassay and Immunochemistry 25 (3(: 241–58. Retrieved 13
December 2012.
Kuhar M, Yamamura HI (Jul 1976(. "Localization of cholinergic muscarinic
receptors in rat brain by light microscopic radioautography". Brain Res. 110 (2(:
229–43.
E. Rutherford and H. Geiger (1908( "An electrical method of counting the number
of α particles from radioactive substances," Proceedings of the Royal Society
(London), Series A, vol. 81, no. 546,
A Handbook of Radioactivity Measurements Procedures, 2nd edition: (Report No.
58), National Council on Radiation Protection and Measurements (NCRP( ,
1985 ISBN 0-913392-71-5,pages 30-31
WWW.GOOGLE.COM/IMAGES
WWW.SLIDESHARE.NET
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