PRESENTED BY

S .VISWANTH REDDY
M.Pharmacy 1stYear(pharmacology)
Gokaraju rangaraju college of pharmacy
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 ENZYME LINKED
IMMUNOSORBENT
ASSAY
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 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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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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 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
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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Materials Needed
Testing sample
Antibody (1st, 2nd) / Antigen
Polystyrene microtiter plate
Blocking buffer
Washing buffer
Substrate
Enzyme

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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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 ANTIBODY ( Ab)
Antibody: proteins produced by the
immune system which help defend
against antigens

SYMBOL FOR
ANTIBODY

Y
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Antibodies (Immunoglobulins)

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Specimen Sample For ELISA
SERUM

CSF

SPUTUM

URINE

SEMEN
SUPERNATANT OF CULUTRE

STOOL
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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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 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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 Secondary
antibody
Substrate
ym e
Enz

Coloured
product

Primary
antibody

Different antigens in sample

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Basic
Steps Of
Enzyme-
Linked
Immunos
orbant
Assay

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TYPES OF ELISA

◦ Indirect elisa

◦ Sandwhich elisa

◦ Competetive elisa

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Indirect elisa

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 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
20
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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 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

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 ( 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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Results

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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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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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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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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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 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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 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

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Limitations

Results may not be absolute •

Antibody must be available •
Concentration may be unclear •
False positive possible •
False negative possible •

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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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 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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Sensitivity of various immunoassays

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 Equipments for performing
the ELISA test

Pipettes
Incubator

ELISA reader

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ELISA READER

(THERMOLAB SYSTEM (USA

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 ]

Radioimmunoassay

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 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.

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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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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]
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 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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 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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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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 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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 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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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

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Antibodies: types of
labelling

,Radio-isotopes-
Enzymes -
Fluorescent
Chemi-luminescent
probes
Metal tags

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 Radioimmunoassay
(RIA)
 Advantages  Disadvantages
◦ Flexibility ◦ Toxicity
◦ Sensitivity ◦ Shelf life
◦ Size ◦ Disposal costs

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 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

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 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).

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53
 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
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INSTRUMENTATION

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 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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r r ∝ [ Ag ]
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