In this slide contains introduction, principle, types, equipment's and applications of Enzyme linked immunosorbent assay. Presented by: D.Sudheer Reddy. (Department of pharmacology) RIPER, anantapur.

1. ELISA(Enzyme Linked ImmunoSorbent Assay)
A Seminar as a part of curricular requirement of M-Pharmacy
Ι year Ι semester
Presented by
D.Sudheer Reddy
Reg.No:20L81S0113
Under the Guidance/Mentorship of
Dr.P.Ramalingam,M.Pharam.,Ph.D.,R&D Director
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Raghavendra Institute of Pharmaceutical Educational and Research- AUTONOMOUS
K.R. Palli Cross, Chiyyedu, Anantapuramu A.P - 515721

2. CONTENTS
•Introduction
•Principle
•Types
•Equipments
•Advantages & Disadvantages
•Applications
•conclusion
•Reference
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3. INTRODUCTION TO ELISA
• ELISA,or enzyme-linked immunosorbet 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 commonly
employed for HIV.It has high sensitivity.
• It is useful & powerful method to estimating ng/mL to pg/Ml ordered materials in
the solution.
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4. Why known as……….?
Enzyme Linked Immunosorbent Assay
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.
5. Plate based immunoassay.
6. Detects and quantifies substances such as peptides,poteins,hormones and
antibodies.
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5. PRINCIPLE:
• ELISA use as an enzyme to detect the binding of antigen (Ag) antibody (Ab).
• The enzyme converts a colorless substrate (chromogen) to a coloured
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 test is designed.
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6. Types of ELISA:
• Frequently there are 3 types of ELISA on the basis of binding structure between
Basis of binding structure between the Antibody and Antigen.
 Direct ELISA
 Indirect ELISA
 Sandwich ELISA
• Specimen samples for ELISA
 Serum
 Cerebrospinal fluid
 Sputum, urine & Supernatant of culture
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8. Indirect ELISA
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•The indirect ELISA detects in the presence of Antibody in a sample.
•Advantage:
-Wide variety of enzyme linked secondary antibodies available.
-Versatile: many primary antibodies can be made in one species and the same labelled
secondary antibody can be used for detection.
-Increased sensitivity : each primary antibody contains several epitopes that can be
bound by the labelled secondary antibody,allowing for signal amplification.
•Disadvantages:
-Cross-reactivity: might occur with the secondary antibody,resulting in non specific
signal.
-An extra incubation step is required in the procedure.

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10. RIPER
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Direct ELISA
•The direct ELISA detects the presence of Antigen in a sample.
• Advantages:
1. Quick methodology since only one antigen is uesd.
2. Cross reactivity of second antibody is eliminated.
• Disadvantages:
1. Immunoreactivity of primary antibody is reduced because is enzyme
linked.
2. Lesser signal amplification than Indirect ELISA.

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12. Sandwich ELISA
•The sandwich ELISA detects the presence of Antigen in a sample.
•Advantages:
1. High specificity because the antigen is specifically captured and detected.
2. Suitable for crude/impure samples as the antigen does not require
purification prior to measurement.
3. Flexible and sensitive, both direct or indirect detection methods can be used.
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14. Enzymes used in ELISA
Horseradish peroxidase (most commonly used)
Alkaline Phosphatase
β-galactosidase
Lactoperoxidase
Tetra Methyl benzidine
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15. RIPER
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16. Result Determination:
A)IgA and IgG tests:
Plot the O.D.result of each reference, except for the negative reference on the
vertical.
axis (Y-Axis) in relation to the number of corresponding units on the horizontal
axis(x-axis).
Using the absorbance value for each sample, determine the corresponding
concentration of antibodies expressed in units/ml from reference curve.
.
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B)IgM Tests:
We can calculate the cut off value A450nm sample/A450nm positive limit
reference
the normalized value of positive reference is 1.
All samples whose value comparised between 0.8 and 1.0 considered dubious
and all samples whose normalized value above 1.0 are considered positive for
IgM antibodies

18. Applications of ELISA
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1. Hormones 7. Vaccine Quality Control
2. Proteins 8. For GMO (Genetically modified
organism
3. Infections 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

19. Conclusion
•It is a good techniques and being popular today because of its simple and not
involve radiation.
•It is easy than other test.
•It is most useful than other test because of their sensitivity.
•It is not harmful.
• Besides its disadvantages the technique is widely used in diagnostics and drug
screening.
•Chromogenic detection method used in ELISA.
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20. Raghavendra Institute of Pharmaceutical Educational and Research- AUTONOMOUS
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Reference
1. Engvall E, Perlmann P.Enzyme linked immunosorbent assay.Quantitative assay
of immunoglobulin G.Immunochemistry.1971;8(9):871-874.
2. Kuhar M,Yamamura HI.Localization of cholinergic muscarinic receptors in rat
brain by light microscopic radioautography.Brain Res.1976;110(2):229-43.
3. Journal of immunoassay and immunochemistry.2012;25(3):241-58.