ELISA (enzyme-linked immunosorbent assay) is a sensitive analytical method for detecting low amounts of substances like proteins, peptides, hormones, and infectious agents. It uses antibodies and enzyme-labeled detection molecules to detect the presence of an antigen or antibody in a sample. There are two main types - sandwich ELISA, which is most sensitive and requires the analyte to have at least two binding sites, and competitive ELISA, which is useful for monovalent analytes. ELISA has advantages like specificity, sensitivity, and ease of use, though limitations include potential for false positives and negatives. It has many applications like detecting hormones, proteins, infectious agents, and more.

1. ELISA
SUBMITTED TO
Mr. Mohan Chavan
Assistant professor pla bio chem.
Dept of AG. Biotechnology
Agricultural College Hassan
BY
MANOJ B.S
BLH 1027
Agricultural College
Hassan

2. Contents
History
Introduction
Principles
Design
Types of ELISA
Application
Conclusion
References

3. HISTORY
The term ELISA was first used by Engvall and
Perlama in 1971.
The ELISA test, was the first screening test
commonly employed for HIV.

4. INTRODUCTION
ELISA means enzyme linked immunosorbent assay
it includes.
Antigen of interest is absorbed on to plastic
surface(sorbent)
Antigen is recognized by specific
antibody(“immuno”)
This antibody is recognized by second antibody
which has Enzyme attached
Substrate reacts with enzyme to produce product,
usually coloured.

5. PRINCIPLE OF ELISA.
Use an enzyme to detect binding of Antigen (Ag)
Antibody (Ab).
The Enzyme converts a colourless substrate(chromogen)
to a coloured product, indicating the presence of Ag:Ab
binding.
An ELISA can be used to detect either the presence of
Antigen or Antibody in a sample depending how the test
is designd.
ELISA was developed in 1970 and became rapidly
accepted.

7. ELISA
• What is an ELISA?
• • Enzyme Linked Immunosorbant Assay
• • Immuno = antibody
• 􀂃 A very sensitive analytical method for measuring/detecting very
low
• amounts of a substance
• • Frequently little or not pretreatment of the sample is required
• 􀂃 A very wide variety of substances can be detected
• • Proteins
• 􀂃 Plant and animal proteins ( hormones, enzymes, cytokines)
• 􀂃 Microbial proteins (viral, bacterial, fungal, bacterial)
• • Other Biomolecules- small to large
• 􀂃 Peptides, Polysaccharides, Gycolipids, Steroids, Organic Toxins,
• Polynucleotides, nucleotides
• 􀂃 Drugs (aminoglycosides, cocaine)

8. ANTIGEN(Ag).
• Any “thing”, foreign to the immune system
.e.g.bacteria,viruses, etc....
• Protein molecules
• Corbohydrate molecule.
• Microorganisms
• Allergens
• Viruses etc….

9. ANTIBODY(Ab)
• Proteins produced by the immune system
which help defend against antigens

10. What do you need to make Antibody
Sandwich Assay
• 􀂃 High affinity and selective Capture Antibody for coating the solid phase
• • Purified antibody for coating
• 􀂃 Solid Phase- Microplate
• 􀂃 Blocker
• • BSA, Casein
• 􀂃 Buffered Solutions- Coating, Washing
• 􀂃 High affinity and selective Antibody for Detection (labeled with enzyme or
• tag (biotin))
• 􀂃 Substrate and Stop solution (Colorimetric, Flurometric, Luminescent)
• 􀂃 Calibrator Solutions- To make the standard curve
• 􀂃 Sample diluent
• 􀂃 Pipettes
• 􀂃 Detection instrument
• 􀂃 Plate Washer
• 􀂃 Plate Sealer tape

11. HOW DO THEY WORK?
• • How do they Work?
• • A subclass solid phase heterogeneous
ligand binding assays
• 􀂃 Solid Phase to facilitate separation label vs
non labeled reagents
• 􀂃 Heterogeneous- they require washing steps
• • Immuno = Antibody-Antigen binding
• 􀂃 Analogous assays can be made using other
binding pairs
• • Receptor or Binding protein—Receptor –
Ligand
• • Strong (high affinity) and specific binding
of Antibody to Antigen enables
• assays to be designed.
• 􀂃 Lock and Key

13. Basic ELISA Types
• Two Basic Designs
• •Non Competitive (Sandwich assay)
• •Solid Phase Antibody or Antigen===Analyte===Labeled Detector Molecule
• •Most frequently type of ELISA used
• •Most sensitive
• •Several steps in protocol
• • Need an analyte with at least two binding sites (bivalent or multivalant)
• •Can not be used for very simple antigens (monovalent).
• •Competitive- EIA (Enzyme Immunoassay)
• •Solid phase antibody or antigen ==== labeled antigen or labeled antibody plus analyte.
• •Solid Phase or labeled analyte competes with analye in the sample for binding to antibody
• •Useful for monovalent analytes
• •Only need one antibody
• •Less steps
• •Less sensitive

17. ADVANTAGESOF ELISA
• Reagents are relatively cheap and have 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.

18. DISADVANDAGES OF ELISA
• 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.

19. LIMITATIONS
• Results may not absolute
• Antibody must be available
• Concentration may be unclear
• False positive possible
• False negative possible

20. APPLICATIONS OF ELISA
• Hormones
• Proteins
• Infectious agent
• Drug markers
• Tumor markers
• Serum proteins
• Vaccine quality control
• Genetically modified organism
• For rapid test
• In clinical research