ELISA & RIA

1. PRESENTED BYPRESENTED BY
S . VISWANTH REDDYS . VISWANTH REDDY
M.Pharmacy 1M.Pharmacy 1
stst
Year(pharmacology)Year(pharmacology)
Gokaraju rangaraju college of pharmacyGokaraju rangaraju college of pharmacy
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2. ENZYME LINKED
IMMUNOSORBENT
ASSAY
2

3. CONTENTSCONTENTS
Introduction to ELISAIntroduction to ELISA
Principle & procedurePrinciple & procedure
Materials neededMaterials needed
Types of ELISATypes of ELISA
Advantages & disadvantages of ELISAAdvantages & disadvantages of ELISA
ApplicationsApplications
ELISAELISA
RIARIA
Introduction to RIAIntroduction to RIA
Principle & procedurePrinciple & procedure
Materials neededMaterials needed
Advantages & disadvantages of RIAAdvantages & disadvantages of RIA
InstrumentationInstrumentation
ApplicationsApplications
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4. INTRODUCTION TO ELISAINTRODUCTION 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 ......?Why known as ......?
EEnzyme nzyme LLinked inked IImmunommunossorbent orbent AAssayssay
1. Antigen of interest is absorbed on to plastic
surface (‘sorbent’(.
2. Antigen is recognised by specific antibody
(‘immuno’(.
3. This antibody is recognised by second antibody
(‘immuno’( which has enzyme attached (‘enzyme-
linked’(.
4. Substrate reacts with enzyme to produce product,
usually coloured.
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6. BASIC PRINCIPLE OF ELISABASIC 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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7. 7

8. Materials NeededMaterials Needed
Testing sample
Antibody (1
st
, 2
nd
) / Antigen
Polystyrene microtiter plate
Blocking buffer
Washing buffer
Substrate
Enzyme
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9. ANTIGEN (Ag)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.
Microorganisms
Allergens.
Viruses Etc.
SYMBOL FOR ANTIGEN
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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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11. Antibodies (ImmunoglobulinsAntibodies (Immunoglobulins))
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12. Specimen Sample For ELISASpecimen Sample For ELISA
SERUM
CSF
SPUTUM
URINE
SEMEN
SUPERNATANT OF CULUTRE
STOOL
12

13. Enzymes Used in ElisaEnzymes 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 SUBSTRATEENZYME SUBSTRATE
Initially the substrate should be colorless
After degradation by the enzyme it
should be strongly colored or fluorescent.
ENZYME SUBSTRATE CHROMOGEN STOPPING
Alkaline
Phosphatase
p-NPP p-NPP+
diethandamine+Mg
Cl2
1 M NaOH
Horse radish
Peroxidase
H2O2 Tetramethylbenzidi
ne + Phosphate –
Citrate buffer
1 M H2SO4
Horse radish
Peroxidase
H2O2 O –
Phenylenediamine
+ HCl
1 M HCl
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15. Substrate
Primary
antibody
Enzyme
Secondary
antibody
Different antigens in sample
Coloured
product
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16. Basic Basic
Steps Of Steps Of
EEnzyme-nzyme-
LLinkedinked
IImmunommunoss
orbantorbant
AAssayssay
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17. TYPES OF ELISATYPES OF ELISA
◦Indirect elisa
◦Sandwhich elisa
◦Competetive elisa
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18. Indirect elisaIndirect elisa
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19. Sandwich elisaSandwich 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. 20

21. ELISA SANDWICH FORMATELISA SANDWICH FORMAT
Y Y Y
Y Y Y
Y
Y
Y
Y Y Y
Y Y Y
2nd antibody with enzyme
Antibody/Antigen
Antibody
Y Y Y
Y Y Y
enzyme produces colour
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22. Competitive ElisaCompetitive 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. Comparison between Indirect Sandwich & Competitive ELISA
to detect Ab (HIV, HCV(
to detect Ag ( Tumor Markers, Hormones (
to detect Ag ( Free Testosterone(
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24. ResultsResults
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25. Importance of incubation step:-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 PlateElisa 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.  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 .
TEST PERFORMANCETEST PERFORMANCE
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28. 28

29. 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

ELISA PLATE READY
FOR READING
29

30. Advantages of ELISAAdvantages 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 ELISADisadvantages 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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32. LimitationsLimitations
•Results may not be absolute
•Antibody must be available
•Concentration may be unclear
•False positive possible
•False negative possible
32

33. APPLICATIONS OF ELISAAPPLICATIONS 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
food allergens, such as milk, peanuts, walnuts, almonds, and eggs
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34. APPLICATIONS OF ELISAAPPLICATIONS OF ELISA
1- Hormones 7- Vaccine Quality Control
2- Proteins 8- FOR GMO (Genetically modified
organism)
3- Infectious Agent ( Viral, Bacterial,
Parasitic, Fungal )
9- For Rapid Test
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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35. Sensitivity of various immunoassays
35

36. Equipments for performing
the ELISA test
Pipettes
Incubator
ELISA reader
36

37. ELISA READERELISA READER
THERMOLAB SYSTEM (USA(
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38. 38

39. Radioimmunoassay Radioimmunoassay
]
39

40.  RadioimmunoassayRadioimmunoassay (RIA) is a very sensitive (RIA) is a very sensitive  in vitroin vitro assay assay technique  technique
used to measure concentrations of antigens (for example, hormone levels in used to measure concentrations of antigens (for example, hormone levels in
theblood) by use of antibodies.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.
INTRODUCTIONINTRODUCTION
40

41. To perform a radioimmunoassay, a known quantity of an antigen is To perform a radioimmunoassay, a known quantity of an antigen is
made radioactive, frequently by labeling it with gamma-made radioactive, frequently by labeling it with gamma-
radioactive isotopes of iodine attached to tyrosine. radioactive isotopes of iodine attached to tyrosine.
This radiolabeled antigen is then mixed with a known amount 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 of antibody for that antigen, and as a result, the two specifically bind to one
another. another.
Then, a sample of serum from a patient containing an unknown quantity of Then, a sample of serum from a patient containing an unknown quantity of
that same antigen is added. that same antigen is added.
This causes the unlabeled (or "cold") antigen from the serum to compete This causes the unlabeled (or "cold") antigen from the serum to compete
with the radiolabeled antigen ("hot") for antibody binding sites. As with the radiolabeled antigen ("hot") for antibody binding sites. As
theconcentration of "cold" antigen is increased, more of it binds to the theconcentration of "cold" antigen is increased, more of it binds to the
antibody, displacing the radiolabeled variant, and reducing the ratio of antibody, displacing the radiolabeled variant, and reducing the ratio of
antibody-bound radiolabeled antigen to free radiolabeled antigen. The antibody-bound radiolabeled antigen to free radiolabeled antigen. The
bound antigens are then separated from the unbound ones, and the bound antigens are then separated from the unbound ones, and the
radioactivity of the free antigen remaining in the supernatant is measured radioactivity of the free antigen remaining in the supernatant is measured
using a gamma counter. using a gamma counter.
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42. Principle of RadioimmunoassayPrinciple 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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43. Advantages & Advantages &
Disadvantages of RIADisadvantages 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 RIARequirements 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 Preparation & Radiolabelling of
the Antigen 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 !
45

46. Preparation of the Specific Preparation of the Specific
AntibodyAntibody
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 SystemDevelopment 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
47

48. Assay ProcedureAssay 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

49. -Radio-isotopes,
-Enzymes
Fluorescent
Chemi-luminescent
probes
Metal tags
Antibodies: types of Antibodies: types of
labellinglabelling
49

50. RRadioadioiimmunommunoaassayssay
(RIA(RIA))
Advantages
◦Flexibility
◦Sensitivity
◦Size
Disadvantages
◦Toxicity
◦Shelf life
◦Disposal costs
50

51. 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
Advantages
51

52. The main drawbacks to radioimmunoassay are
the expense and hazards of preparing and
handling the radioactive antigen.
Both
125
I or
131
I 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).
limitations
52

53. 53

54. INSTRUMENTATIONINSTRUMENTATION
Incubate tissue with
radioactive ligand
Expose to film
or emulsion
Isotope will emit
radiation (usually beta(
Radiation will hit silver grains in emulsion and expose them
Autoradiography
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55. INSTRUMENTATIONINSTRUMENTATION
55

56. REFERENCESREFERENCES
 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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