elisa Applications methods types The enzyme-linked immuno sorbent assay (ELISA) is an assay technique designed for detecting and quantifying peptides, proteins, antibodies and hormones. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality-control check in various industries. The process of the ELISA result in a colored end product which correlates to the amount of analyte present in the original sample.

1. SJM College of Pharmacy,
Chitradurga
Prepare By,
AdarshaBP
Ass Professor(Pharmacognosy)
SJM College of Pharmacy
1
PHARMACEUTICAL
BIOTECHNOLOGY

2. ELISA
• The enzyme-linked immuno sorbent assay (ELISA) is an assay technique
designed for detecting and quantifying peptides, proteins, antibodies and
hormones.
• The ELISA has been used as a diagnostic tool in medicine and plant
pathology, as well as a quality-control check in various industries.
• The process of the ELISA result in a colored end product which correlates
to the amount of analyte present in the original sample.

3. • ELISAs are typically performed in 96 well (or 384 well)
polystyrene plates.

4. Principle
• Principle of Enzyme-linked Immunosorbent Assays
(ELISAs) is based on the specific interaction between
antibody and antigen.
• The ELISA can be used to detect antigens that are
recognized by an antibody or it can be used to detect
antibodies that are recognized by an antigen.

5. Antibodies for ELISA
• All ELISA formats rely on the specific
interaction between an antigen and a matching
antibody.
• The antibodies used in an ELISA can be either:
• monoclonal (capable of specific binding to a single
unique epitope)
• OR
• polyclonal (capable of binding to multiple
epitopes).

6. Probes for ELISA
• Radioactive isotopes, enzymes and fluorescent
dyes are different types of chemical tags that
have been conjugated to secondary or primary
antibodies(probes) to make probes detectable.
• Horseradish peroxidase (HRP) is most commonly
used for blotting, immunoassays and it converts
its substrate ABTS (2,2'-Azinobis [3-
ethylbenzothiazoline- 6-sulfonic acid]-
diammonium salt) in green end product.

7. Blocking & Washing Buffers
• A blocking buffer is a solution of irrelevant protein, with a
small percentage of detergent. Blocking buffer block the
places where target proteins have not been attached.
• Washing buffer are used to remove nonspecifically bound
and unbound reagents.
• Washing is performed by buffers such as Tris-buffered saline (TBS) or
phosphate-buffered saline (PBS) with detergent such as 0.05% Tween-
20.

8. Types of ELISA
• INDIRECT ELISA
• DIRECT ELISA
• SANDWICH ELISA
• COMPETETIVE ELISA
NON -COMPETETIVE
ELISA

9. IndirectELISA
• Antigen is added to plate.
• Added Blocking buffer.
• Suitable primary antibody is added.
• Secondary antibody- HRPO is then
added which recognizes and binds to
primary antibody.
• TMBsubstrate is added, is converted
to detectable form.

10. Advantages of Indirect Detection
• Wide variety of labeled secondary antibodies are available commercially.
• Sensitivity is increased because each primary antibody contains several
epitopes that can be bound by the labeled secondary antibody, allowing for
signal amplification.
Disadvantages of Indirect Detection
• Cross-reactivity may occur with the secondary antibody, resulting in
nonspecific signal.
• An extra incubation step is required in the procedure.

11. Direct ELISA
• Apply a sample of known antigen to a surface.
• Enzyme linked primary antibody is applied to
the plate.
• Wash the plate. After this wash, only the
antibody-antigen complexes remain
attached.
• Apply a substrate which is converted by
the enzyme to elicit a chromogenic signal.

12. Advantages of Direct Detection
• Quick methodology since only one antibody is used.
• Cross-reactivity of secondary antibody is eliminated.
Disadvantages of Direct Detection
• Labeling of every primary antibody is time-consuming and expensive.
• Little signal amplification.

13. Sandwich ELISA
• Plate is coated with suitable antibody.
• Blocking buffer is added.
• Sample is added to plate so antigen is bounded by capture antibody.
• A suitable biotin labeled detection antibody is added to plate.
• Enzyme HRPO is added and binds the biotin labeled detection
antibody.
• TMB substrate is added and converted by HRPO to colored product.

14. Competitive ELISA
• Solid phase coated
with antibody
• Add unknown amount of
unlabeled antigen and known
amount of labeled antigen
• Free and labeled antigen are
captured
• Color formation by oxidation of
substrate into a colored
compound

15. Advantages
• Suitable for complex samples, since the antigen does
not require purification prior to measurement.
Disadvantages
• Each antigen may require a different method to couple
it to the enzyme.

16. • Presence of antigen or the presence of antibody in a sample can be detected by ELISA.
• ELISA can be used to determination of serum antibody concentrations in a virus test (such
as HIV test).
• ELISA can also be used in home pregnancy test.
• It is used in food industry to detect potential food allergens such as milk, peanuts, walnuts, almonds,
and eggs.
• ELISA can also be used in toxicology as a rapid presumptive screen for certain classes of drugs.
Applications of ELISA